Chiral Mutagenesis of Insulin. Foldability and Function Are Inversely Regulated by a Stereospecific Switch in the B Chain^,

Abstract

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding D and L amino acid substitutions in the hormoneto define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly B8 lies within a -turn and exhibits a positive ϕ angle (like a D amino acid); in the alternative R state, Gly B8 is part of an -helix and exhibits a negative ϕ angle (like an L amino acid). Respective B chain libraries containing mixtures of D or L substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: L amino acids impede native disulfide pairing, whereas diverse D substitutions are well-tolerated. Strikingly, D substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, L analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of D and L analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 ϕ angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated † This work was supported in part by Diabetes Research and Training Center at the University of Chicago (S. H SUPPORTING INFORMATION AVAILABLENine figures illustrating disulfide pairing and structural relationships in insulin crystals, visible absorption spectra of cobalt-substituted hexamers, additional CD and NMR spectra, diagonal plot of NOEs, and summary of NMR sequential assignment. Nine tables providing B8 dihedral angles, summary of mutations at sites neighboring B8, crystallographic unit-cell dimensions, NMR resonance assignments, statistical information pertaining to DG/RMD ensemble, and restraints. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2013 December 02. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assemb...

Insulin analog with additional disulfide bond has increased stability and preserved activity

“…Dihedral angles of GlyB8 range from positive in the T-state (56.4 6 4.1) to negative in the R-state (À63.0 6 3.12). 32 Dihedral angles of GlyB8 in 4SS-insulin were found to be positive and similar to those observed in the T-state (46.2 and 42.4 for the two molecules in the asymmetric unit). Furthermore, the CD spectroscopy results show no changes in CD spectra upon phenol titration, suggesting no or only very limited changes in the ''hinge'' region.…”

“…Previously, it was also suggested that reorganization of the Nterminal end of the B-chain, such as the transition from T-state to R-state, is necessary for the insulin receptor binding. 32 Our results demonstrate for the first time that structural flexibility of the N-terminal end of the B-chain of insulin is not a prerequisite for binding and that an insulin analog not capable of forming the classical R-state is fully active. Since the first four amino acids of the B-chain do not have a large affect on insulin interaction with the insulin receptor, 42,43 the ''functional'' difference between the T-state and the R-state lies around the GlyB8 ''hinge'' region.…”

“…The B8 U dihedral angles in HI are found to be positive in the T-state (56.4 6 4.1) and negative in the R-state (À63.0 6 3.12). 32 Four hydrogen bonds are formed between residues B1-A13, B3-A11, and A9-B5 [ Fig. 1(D)].…”

Insulin gene mutations as a cause of permanent neonatal diabetes

“…Because B8 glycine has a positive (Phi) dihedral angle (forbidden for L-amino acids), the presence of L-serine at B8 requires a reorientation of B8 conformation to accommodate its side chain, which results in rotation of B1-B8, turning the B7 cysteine away from its intended partner at A7. This change is reflected in lowered thermodynamic stability of the insulin molecule and significantly impairs yields in chain recombination experiments (24,25). On the basis of these and other observations, Nakagawa et al (24) have suggested that ''the correct B8 conformation plays a critical role in the mechanism of disulfide pairing'' during insulin biosynthesis and when substituted may ''kinetically block the formation of the A7-B7 disulfide bridge.''…”

“…This change is reflected in lowered thermodynamic stability of the insulin molecule and significantly impairs yields in chain recombination experiments (24,25). On the basis of these and other observations, Nakagawa et al (24) have suggested that ''the correct B8 conformation plays a critical role in the mechanism of disulfide pairing'' during insulin biosynthesis and when substituted may ''kinetically block the formation of the A7-B7 disulfide bridge.'' Similar considerations apply to B23 glycine where L-amino acid substitutions would likely disrupt normal folding of the peptide chain near the critical A20-B19 disulfide bridge (26).…”